Prevalence of extended-spectrum beta-lactamase genes in Acinetobacter baumannii strains isolated from nosocomial infections in Tehran, Iran

Background and objectives: blaSHV, blaTEM and blaVEB are a group of Extended-Spectrum Beta-Lactamase enzymes (ESBLs) which are able to hydrolyze Penicillins and some cephalosporin antibiotics. The present study evaluated the frequency of ESBL genes blaSHV, blaTEM and blaVEB in Acinetobacter baumannii strains isolated from nosocomial infections to outline the importance of these genes in antibiotic resistance. Methods: One hundred Acinetobacter baumannii strains were isolated from different nosocomial infections. After antibiotic resistance evaluation with the Kirby-Bauer disc-diffusion method, the Minimum Inhibitory Concentration (MIC) of Ciprofloxacin was measured using the E-test method. Then, the ESBL producing strains were identified employing Combined Disk Methods. Finally, all isolates were evaluated with the Polymerase Chain Reaction (PCR) technique to detect the ESBL genes of interest. Results: Out of 100 Acinetobacter baumannii isolates, 59% were ESBL positive according to the phenotypic method. The PCR assay could not detect the blaSHV and blaVEB genes in the studied isolates, but the presence of blaTEM gene was demonstrated in 42% of the strains. Conclusion: The high resistance to most antibiotics, the high prevalence of ESBLs-producing strains and also a high prevalence of blaTEM gene in A. baumannii strains found in the current study gives cause for major concern about nosocomial infections in Iran because of the treatment complexity of these strains. Our results highlight the need for infection control measures to prevent the spread of resistant isolates, especially in hospitals.


Introduction
Acinetobacter species are aerobic gram-negative microorganisms responsible for various types of infections such as pneumonia, urinary tract infection and septicemia [1]. Acinetobacter baumannii (A. baumannii) is an important opportunistic pathogen that has caused global outbreaks of nosocomial infections [2].The Acenitobacter infections have been recognized as an emerging problem (one of major cause of nosocomial infections) and appeared to be associated with high mortality rates throughout the world [3]. The infections caused by Acinetobacter pathogens are often treated with Cephalosporins, including Ceftazidime, ceftriaxone, aminoglycosides such as tobramycin and amikacin, Carbapenems, and tetracycline. To date, however, most strains of A. baumannii have become increasingly resistant to almost all these currently available antibacterial agents due to the presence of mobile genetic elements, such as insertion sequences (ISs), plasmids, integrons and resistance islands [1], [2]. These mobile elements carry and transfer antimicrobial resistance genes to another bacterium and easily spread among various species and even genera of microorganisms. Furthermore, the transfer of these elements between chromosome and plasmid may facilitate the rapid spread of the resistance genes among different strains of bacteria. An increasing drug-resistance rate among A. baumannii strains is a major concern in hospitals worldwide [4], [5], [6]. The antimicrobial resistance in this nosocomial pathogen is mainly caused by beta-lactamase (βlactamases) inactivating enzymes, alteration of membrane porin channels, and mutations that change cellular functions [5]. However, the most common mechanism of resistance is the production of hydrolytic enzymes of antimicrobial agents, including extended-spectrum betalactamases (ESBLs) that belong to Ambler classes A, D and B [7], [8], [9], [10]. ESBL-producing strains are mutant, acquired plasmid-mediated β-lactamases which demonstrate unique hydrolytic properties. They are enzymes with the potential to digest β-lactamase antibiotics, which possess Oxyimino groups such as Oxyimino-Cephalosporins. However, they are inhibited by Clavulanate and Tazobactam, β-lactamase inhibitors [4]. Clinical types of ESBL genes includes bla SHV , bla TEM , bla VEB , bla KPC , bla PER , bla BEL-1 , bla BES-1 , bla SFO-1 , bla TLA and bla BIC that are associated with mobile genetic elements, predominantly plasmids [11]. The wide use of numerous new β-lactam antibiotics in recent decades has led to the emergence of ESBLs, which are mostly derivatives of TEM-1 (named for the patient, Temoneira) and SHV-1 (sulfhydryl variable) enzymes. They are capable of hydrolyzing a wide range of β-lactam antibiotics, except carbapenems that have been detected in Acinetobacter strains and many gram-negative bacteria [12]. Non-TEM-, non-SHV-derived ESBLs (VEB [Vietnamese extended-spectrum-beta-lactamase]) have been documented in Acinetobacter species from Europe and Asia but not yet from the Americas [11], [13]. The bla VEB-1 ESBL gene is belonging to class A β-lactamase and is located in a class 1 integron. These genetic structures are responsible for the expression of cassette-associated and mobile resistance genes, initially detected in Enterobacteriaceae and Pseudomonas aeruginosa from Southeast Asia [14], [15]. Subsequently, it has been described in clonally related A. baumannii isolates recovered during an outbreak that lasted 9 months (August 2001-April 2002) in the intensive care unit of a hospital in northern France. In these strains, the location of the bla VEB-1 gene on the chromosomes and integrons was identified [15]. This enzyme is associated with a high level of resistant to Cephems, Monobactams and Ceftazidime [16], [17]. In light of the above, the increasing rate of antibiotic consumption and its impact on treatment failure due to misuse or overuse of antibiotics by patients, and finally widespread drug-resistant strains particularly in hospitals, it is imperative to search for new means of controlling the increasing mortality rate based on the failure of drug therapy [18], [19], [20]. Regarding the growing importance of ESBLs in antibiotic resistance and its impact on treatment failure, this study was performed to evaluate the antimicrobial susceptibility patterns of clinical strains and determine the frequency of bla SHV , bla TEM and bla VEB genes in A. baumannii strains isolated from several hospitals in Tehran.

Bacterial strains
In this cross-sectional descriptive study, 100 isolated samples of A. baumannii were collected from 10 hospitals in Tehran (Imam Hoseyn, Mofid, Imam khomeyni, Shahid Labafi nazhad, Loghman, Khatamalanbya, Milad, Mostafa Khomeyni, Shariati and Motahari) from October 2014 to April 2015. The specimens were obtained from wounds, tracheae, blood, sputum, catheters, pleural fluid, urine and CSF of hospitalized patients. These isolates were collected from different hospital wards, including ICU, Burns, Internal Medicine, Emergency Medicine, Surgery, Medical, Neurological, BMT, CCU, Orthopedic and Maxillofacial Surgery wards. Replicated isolates from the same patients were excluded from the study. All isolates were identified morphologically and using conventional biochemical methods. The following phenotypic and bacteriological tests were used in this study: Gram staining, colony morphologies, McConkey's agar, TSI, oxidase reaction, lysine decarboxylase, growth at 37°C and 45°C, hydrolyzed gelatin, citrate utilization, OF, and hemolysis on blood agar with 5% sheep blood. The obtained strains were preserved in tryptic soy broth (Merck, Darmstadt, Germany) containing 15% glycerol [21].

Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) test was performed according to CLSI guidelines by the E-test method for Ciprofloxacin-resistant isolates on Mueller-Hinton agar medium. E-test-Imipenem strips were applied on the plates, and the plates were incubated at 37°C in air for 16 to 20 h [23]. Non-susceptible A. baumannii strains with MIC≥32 µg/ml were considered as Ciprofloxacin resistant [24]. A. baumannii ATCC19606 and Pseudomonas aeruginosa ATCC27853 served as controls for the Ciprofloxacin E-test method.

Phenotypic detection of ESBL-producing isolates
To detect ESBLs, all the isolates were tested employing the disk diffusion test (CDDT) containing Ceftazidime (CAZ) 30 µg and Cefotaxime (CTX) 30 µg with a combination of CAZ 30 µg+clavulanic acid (CA) 10 µg and CTX 30 µg+CA 10 µg per disc (Mast Group, Merseyside, UK). Zones of inhibition were compared with the CTX and CAZ discs alone and compared with the combined CAZ 30 µg+CA 10 µg and CTX 30 µg+CA 10 µg discs. An increase in zone diameter of ≥5 mm in the presence of clavulanic acid indicated the existence of ESBL in the test microorganism. Escherichia coli ATCC25922 and Klebsiella pneumonia ATCC700603 were used as negative and positive controls for ESBL production, respectively [25].

DNA extraction
For molecular diagnosis, the total DNA of all A. baumannii isolates was extracted by the DNA extraction kit (Bioneer Company, Korea, Cat. number K-3032-2) specifically for the given bacterial colony.

Detection of ESBL genes by PCR method
The genetic basis of the β-lactam resistance mediated by enzymatic mechanisms was investigated using the PCR method on all of the isolates phenotypically positive for ESBL. The PCR assay for detection of β-lactamase genes bla SHV , bla TEM and bla VEB was carried out using the previously reported specific oligonucleotide primers shown in Table 1. The PCR mixture contained the DNA template, Forward/Reverse primers, and master mix (Bioneer Co., Korea, Cat. number K-2016). Amplification was carried out with the following thermocycling conditions. The cycling parameters used for the bla SHV gene were: 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 30 sec and a final extension at 72°C for 10 min. The parameters for the bla TEM gene were: 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 59°C for 1 min, extension at 72°C for 1 min and a final extension at 72°C for 10 min. The parameters used for the bla VEB

Results
Among 100 collected strains, 57 and 43 were isolated from men and women, respectively. The majority and minority of isolates were obtained from wounds (40%) and CSF (3%), respectively ( Table 2). The highest isolation rate was associated with the ICU ward and the lowest with the orthopedic ward. As shown in Figure 1, sensitivity to Meropenem, Gatifloxacin and Levofloxacin was 29%, 11% and 10% respectively, which were most effective antibiotics against clinical isolates. The maximum rate of resistance was related to Ticarcilin-Clavelonic acid. The E-test illustrated that the MIC level of ciprofloxacin in all ciprofloxacin-resistant A. baumannii isolates (91%) was ≥32 µg/ml. In the phenotypic method, 59 (59%) isolates produced ESBL enzymes (increase of ≥7 mm in zone diameter of CAZ-clavulanic acid disks compared to CAZ disks. same value as for the CTX+clavulanic acid compared to Imipenem disks). In the molecular assay (PCR), bla SHV and bla VEB genes were not detected in any A. baumannii clinical isolates, but the presence of the bla TEM gene was demonstrated in 42% of these strains (Figure 2, Figure 3 and Figure 4).

Discussion
A. baumannii is responsible for hospital-acquired infections and has recently become one of the most important healthcare-associated infections in hospitals. Infections caused by this pathogen often leads to significant mortality and morbidity [26]. Antimicrobial resistance in A. baumannii has become a worldwide problem, especially in healthcare centers and hospitalized patients. The emergence of clinical A. baumannii isolates with diverse antibiotic resistance phenotypes leads to difficulties in treating infections caused by this pathogen [3], [27]. In recent years, A. baumannii has been commonly reported as multiple-drug resistant (MDR); the resistance rates to Imipenem, Meropenem, Ceftazidime, Piperacillin/Tazobactam, Ciprofloxacin and Gentamicin in Latin America seem to be among the world's largest [28]. In the present study, resistance to Meropenem, Gatifloxacin, Levofloxacin, Ceftazidim, Pipracilin-Tazobactam, Cotrimoxazole and Ticarcilin-Clavelonic acid were 71%, 89%, 90%, 93%,   [32], [33], [34], [35]. In our study, of 59 (59%) ESBL-positive A. baumannii strains tested by PCR method, the bla TEM gene was detected in 42% of them, while bla SHV and bla VEB genes were not found in these isolates. In a study conducted by Fallah et al., 84.2% of the investigated A. baumannii strains were ESBL positive according to the combined disk diffusion test; the bla VEB gene was found in 39.5% of these isolates [11]. In the study carried out by Pasterán et al. (2006) that investigated 21 ESBL-producing A. baumannii strains, the bla VEB gene was presented in 10 (47.6%) isolates [13]. In a study conducted by Thapa et al., out of 37 A. baumannii clinical isolates, the bla VEB was detected in 7 (9%) strains [14].  [29]. Moreover, in the study by Lopes et al., the PCR technique performed to detect beta-lactamase genes in 50 isolates showed that all isolates harbored the bla TEM gene, while bla SHV and bla VEB genes were not found in any of the strains [38]. The results of these studies regarding the prevalence of blaT EM , blaT EM and bla VEB ESBL genes were similar to our results. This indicates that the prevalence of bla TEM gene is higher than that of bla VEB and bla TEM genes in different regions. In a study by Koo et al., 35 MDR A. baumannii isolates were examined, and 7 antibiotic resistance gene determinants were investigated. They were unable to detect bla TEM , bla SHV and bla VEB beta-lactamase genes [39]. ESBL genes are commonly located on class 1 integrons and are mostly plasmid mediated, and thus transfer easily to other bacteria. This genetic transfer among bacteria causes more rapid spread of ESBL-producing strains. Therefore, the results of different studies can vary due to different regions in which the studies were performed, varied prevalence of pathogenic strains carrying resistance genes especially in hospitals, abusing/overusing of antimicrobial drugs by patients, and study design in term of methodology and sample size [5].

Conclusions
The results of this study reveal an alarming percentage of ESBL-producing strains occurring in Iran. ESBL-producing isolates have emerged as a major challenge, arising from overuse of expanded-spectrum Cephalosporins in hospitals and nursing homes. The high level of resistance to most antibiotics and high prevalence of bla TEM genes in this study indicate the trend to increasing antibiotic resistance in our country and the complexity of treating infections caused by this organism. This study helps understand both the need for more caution in antibiotic consumption and the alarming rate of resistance. Future studies focus on investigation of other ESBL genes. It is necessary to seek a means of monitoring the ESBLs in healthcare settings to prevent the spread of resistant strains and facilitate the selection of appropriate antibiotics for patient treatment.